Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

Circulating miR-221/222 reduces CD4+ T cells by inhibiting CD4 expression in colorectal cancer.

Many patients with cancers have low levels of CD4+ in their peripheral blood. However, the molecular mechanism is still unclear. Here, we found that the blood levels of miR-221 and miR-222 were dramatically increased in patients with colorectal cancer (CRC), and both circulating miR-211 and miR-222 served as sensitive diagnostic markers with an area under the curve of 0.8790 and 0.9148, respectively. Transfection of either miR-221 or miR-222 resulted in the reduction of the surface CD4 antigen level but not the surface CD8 antigen level. The luciferase reporter assay showed that miR-221/222 directly regulated CD4 expression in human primary T cells. These data showed that miR-221/222 levels were upregulated in the blood of patients with CRC and that the expression of CD4 in human primary T cells was inhibited by miR-221/222. These findings provide a novel strategy for modulating the number of CD4+ T cells in the blood and further adjusting the microenvironment suitable for immunotherapy.

PUMILIO proteins promote colorectal cancer growth via suppressing p21.

PUMILIO (PUM) proteins belong to the highly conserved PUF family post-transcriptional regulators involved in diverse biological processes. However, their function in carcinogenesis remains under-explored. Here, we report that Pum1 and Pum2 display increased expression in human colorectal cancer (CRC). Intestine-specific knockout of Pum1 and Pum2 in mice significantly inhibits the progression of colitis-associated cancer in the AOM/DSS model. Knockout or knockdown of Pum1 and/or Pum2 in human CRC cells result in a significant decrease in the tumorigenicity and delayed G1/S transition. We identify p21/Cdkn1a as a direct target of PUM1. Abrogation of the PUM1 binding site in the p21 mRNA also results in decreased cancer cell growth and delayed G1/S transition. Furthermore, intravenous injection of nanoparticle-encapsulated anti-Pum1 and Pum2 siRNAs reduces colorectal tumor growth in murine orthotopic colon cancer models. These findings reveal the requirement of PUM proteins for CRC progression and their potential as therapeutic targets.

Transplanting cells from old but not young donors causes physical dysfunction in older recipients.

Adipose-derived mesenchymal stem cell (ADSC)-based regenerative therapies have shown potential for use in many chronic diseases. Aging diminishes stem cell regenerative potential, yet it is unknown whether stem cells from aged donors cause adverse effects in recipients. ADSCs can be obtained using minimally invasive approaches and possess low immunogenicity. Nevertheless, we found that transplanting ADSCs from old donors, but not those from young donors, induces physical dysfunction in older recipient mice. Using single-cell transcriptomic analysis, we identified a naturally occurring senescent cell-like population in ADSCs primarily from old donors that resembles in vitro-generated senescent cells with regard to a number of key pathways. Our study reveals a previously unrecognized health concern due to ADSCs from old donors and lays the foundation for a new avenue of research to devise interventions to reduce harmful effects of ADSCs from old donors.

miR-221/222 activate the Wnt/ß-catenin signaling to promote triple-negative breast cancer.

Triple-negative breast cancer (TNBC), characterized by the lack of expression of the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor 2, is an aggressive form of cancer that conveys unpredictable and poor prognosis due to limited treatment options and lack of effective targeted therapies. Wnt/ß-catenin signaling is hyperactivated in TNBC, which promotes the progression of TNBC. However, the molecular mechanism of Wnt/ß-catenin activation in TNBC remains unknown. Here, we report the drastic overexpression of miR-221/222 in all of four TNBC cell lines and TNBC primary tumor samples from patients. Furthermore, we demonstrate by both ex vivo and xenograft experiments that inhibiting miR-221/222 expression in a TNBC cell line (MDA-MB-231) suppresses its proliferation, viability, epithelial-to-mesenchymal transition, and migration; whereas expressing miR-221/222 in a non-TNBC line (MCF7) promotes all of the above cancer properties. miR-221/222 achieve so by directly repressing multiple negative regulators of the Wnt/ß-catenin signaling pathway, including WIF1, SFRP2, DKK2, and AXIN2, to activate the pathway. Notably, the level of miR-221/222 expression is inversely correlated whereas that of WIF1, DKK2, SFRP2, and AXIN2 expression is positively correlated with the patient survival. Last, we show that anti-miR-221/222 significantly increases apoptotic cells with tamoxifen/Wnt3a treatment but not with cyclophosphamide/Wnt3a treatment. These results demonstrate that miR-221/222 activate the Wnt/ß-catenin signaling to promote the aggressiveness and TNBC properties of breast cancers, and thus reveal a new prospect for TNBC treatment.

Detection of thrombin with an aptamer-based macromolecule biosensor using bacterial ghost system.

A rapid on-site detection of exogenous proteins without the need for equipped laboratories or skilled personnel would benefit many areas. We built a rapid protein detection platform based on aptamer-induced inner-membrane scaffolds dimerization by virtue of bacterial ghost system. When the detection platform was coincubated with two kinds of aptamers targeting two different sites of thrombin, green fluorescence or ß-lactamase activity were yielded with two different designs. The latter was detected by commercially available testing strips.